The present invention was granted a foreign filing license issued by the Ministero dello sviluppo economico UIBM, on the 30 Apr. 2010, Protocol number 40718.
The present invention relates to a new Ena/VASP protein isoform, uses thereof, diagnostic methods and kits comprising the same.
Carcinogenesis and progression of a tumour lesion are characterized by different gene expression patterns. These expression patterns are defined by different splice-variants and in particular, alternative splicing events frequently affect genes controlling the cytoskeleton organization in tumour cells (Wang G S, Cooper T A. 2007; Gardina P J et al. 2006).
It has been reported that regulatory pathways controlling actin cytoskeleton dynamics are deregulated in cancer cells, and altered expression of proteins controlling actin polymerization frequently occurs affecting growth, survival and migration of tumor cells (Olson M F, Sahai E. Clin Exp Metastasis. 2009).
Unfortunately, despite the progress related to the understanding of molecular mechanisms underlying the carcinogenesis, there are no molecular markers or pathologic prognostic criteria capable to predict the clinical evolution of a tumour lesion in human patient.
Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) are key regulatory molecules controlling cell shape, movement and in particular, actin organization. These proteins target the F-actin network and play an important role in cell migration in a number of cell types and organisms.
Ena/VASP proteins are characterized by a highly conserved N-ter EVH1 domain mediating intracellular localization, a central proline rich domain interacting with the G-actin binding protein profillin, SH3 and WW domains of signalling proteins and a C-Terminal EVH2 domain by which the Ena/VASP proteins tetramerize which also possess the functional F-actin binding domain.
Ena/VASP proteins are involved in actin based movement of cells such as fibroblasts (Bear J E et al. 2002) as well as in cell adhesion (Scott J A, et al. 2006).
Ena/VASP proteins can regulate distinct modes of actin organization at cadherin-adhesive contacts and a fine regulation of their expression is necessary for a correct actin network.
Mena is a member of Ena/VASP family which includes Mena, VASP and Evl, all key regulators of actin dynamics (Krause M, et al. 2003). The analysis of the antibody repertoire specific for the molecules expressed by her autologous tumor of a long surviving breast cancer patient have allowed the isolation of the human ortholog of Mena (hMena) as a tumor antigen stimulating an antibody response in cancer patient and not in healthy donors (Di Modugno et al 2004).
Both in human (Urbanelli L et al. 2006) and in murine models (Gertler F et al. 1996), various Mena splice variants have been described and sequenced and some have been associated with particular tissue expression pattern. In mouse a neuronal (N-Mena) form has been described which possess a longer exon 6 that at protein level includes a larger pro-rich region (Gertler F et al. 1996), whereas a splice variant deriving isoform, lacking the pro-rich region has been reported as spleen specific (Tani K et al. 2003).
Mena is up regulated in mouse and rat invasive breast cancer cells (Wang et al. 2004) and overexpressed in human breast cancer tissues where it represents an early marker of breast cancerogenesis (Di Modugno et al. 2006).
The approach of in vivo invasion assay has recently allowed the identification of invasive specific splice variants in murine and rat tumour cells (Goswami S et al. 2009; Philippar et al 2008).
In humans the molecular cloning of hMena and of a splice variant hMena11a from a luminal breast cancer cell line has previously been reported[u1]. This hMena+11a isoform includes an additional 21.aa peptide in the EVH2 domain of the protein, is phosphorylated following EGF and NRG1 treatment and characterizes the epithelial phenotype of breast cancer cell lines (Di Modugno F et al. 2007).
hMena11a isoform expression characterizes pancreatic cancer cell lines showing the epithelial marker E-Cadherin expression, EGFR dependency and sensibility to Erlotinib treatment (Pino M S et al. 2008; and patent WO2009150494). This isoform expression has been recently reported to be regulated by Epithelial Splicing Regulatory Proteins 1 and 2 (ESRP1 and ESRP2), coordinators of an epithelial cell-type-specific splicing program (Warzecha C C et al. 2009).
It has been reported, in a murine and rat model, that invasive tumor cells express higher levels of Mena mRNA containing the +++ exon, but lack the hMena11a isoform, with respect to the non-invasive tumor cells, but the two isoforms are not alternatively expressed (Goswami et al., 2008). Given the above, it is clear that it would be useful to further clarify the molecular pathway underlying the phenotypic changes that define early markers of cancerogenesis and progression of the neoplastic lesion in human in order to identify markers allowing an early and reliable prediction of the behaviour of a neoplastic lesion or even of a pre neoplastic lesion. Such markers would also allow the physician to identify the most appropriate therapeutic approach. In fact, the possibility of predicting in a patient, at early stages of the pre neoplastic or neoplastic lesion, the clinical evolution thereof, may facilitate the design of the most suitable and effective therapeutic approach.